The present invention generally relates to processing liquid samples containing an analyte, and more specifically to processing biological samples containing a biological analyte, with two or more components, using an in-line flow cell.
Blood tests, such as immunoassay or high performance liquid chromatography (HPLC), require analysis of the components of hemoglobin in a sample. Results of such tests can be expressed as a ratio of the amount of one component (e.g., hemoglobin A1c) of hemoglobin to the amount of a group of components (i.e., group amount). These ratios can be predictive of the presence and management of various diseases and physiological conditions, such as diabetes.
The group amount may differ from a total amount of components because of the exclusion of some components of hemoglobin, such as fetal hemoglobin and hemoglobin variants. The difference between the group amount and total amount is generally small, so as a first pass the total amount can be used as a good approximation for group the amount.
Problems with testing consistency can occur when the total response differs from sample to sample. Solutions have been proposed regarding this problem, as shown in co-assigned U.S. patent application Ser. No. 12/196,132. One current method is to maintain strict control of the total amount of hemoglobin by diluting a sample to a fixed concentration and then testing a fixed volume of the diluted sample, thereby providing a fixed total amount of hemoglobin.
Optimum results have not been achieved thus far, partially due to variability in the total/group amount of hemoglobin in an original sample, settling of the blood cells in the sample, and difficulties in accurately controlling the dilution ratio, as the dilution ratio will have a tolerance range. The first two contributors (i.e., variability and settling) result in sampled hemoglobin concentrations ranging from 90 to 360 grams/liter which give a normalized range of 1-4. Inaccuracies in controlling the dilution ratio can extend this range to 1-5. In a particular HPLC application, this 1-5 range corresponds to total chromatographic peak areas ranging from 1-5 AU-seconds, with a mid value of 3 AU-seconds. Quantification accuracy is compromised as this area deviates from the mid-value. At the extremes of the range, the sample may need to be re-run at a different dilution that results in an area closer to the mid-range. Accordingly, new methods for providing precise and reliable quantification of the components of hemoglobin, as well as other analytes, are desired.